LIVING WITH IT WORKING WITH IT TREATING IT
Background: Diagnosing Invasive Aspergillosis (IA) at an early, curable stage is challenging. Rapid diagnosis is critical to ensure patients receive effective antifungal treatment prior to systemic spread. Polymerase Chain Reaction (PCR) provides a rapid, accurate diagnostic test directly from patient samples.
Materials/methods: Prior to evaluating commercial CE-IVD Aspergillus PCR assays,optimisation of DNA extraction was performed by comparing DNA extraction from spore and hyphae suspensions of Aspergillus fumigatus. Two bead types (Septifast beads and green beads) were analysed, and subjected to varied bead-beating protocols ranging durations of 45-120 seconds and speeds of 3500-7000rpm on the MagNA Lyser (Roche, U.K.). Suspensions were made in simulated sample comprising molecular grade water with carrier RNA, and known negative clinical samples of human serum and broncho-alveolar lavage (BAL). Nucleic acid was extracted on the MagNA Pure Compact (Roche, U.K.) using the MagNA Pure Compact Nucleic Acid Isolation Kit 1 (Roche, U.K.). PCRs were performed with an in-house primer and probe mix, using the Rotor-Gene Q (Qiagen, U.K.).
Results: A. fumigatus DNA was recovered from spore and hyphae suspensions in simulated and clinical samples, however PCR Ct results from hyphae demonstrated a significantly greater recovery yield than spores (P<0.05, paired t-test). Septifast beads demonstrated improved recovery overall with less variation in range compared with Green beads, however neither bead-type recovered DNA from the lowest concentration of spores. The bead-beating time and speed combination that demonstrated greatest DNA recovery was 2 minutes at 3500 rpm which showed a good level of reproducibility between triplicate results. Recovery from clinical samples confirmed no inhibition or adverse effects on DNA elution.
Conclusions: Optimisation of Aspergillus fumigatus DNA extraction from simulated and clinical samples confirmed that recovery is possible when bead-beating is incorporated, however bead type, speed and duration all impact the quantity recovered. DNA was most reliably extracted from hyphae suspensions, and further enhanced when bead-beated using Septifast beads with a bead-beating procedure of 2 minutes at 3500 rpm. This extraction protocol will be employed in subsequent verification work comparing in-house and commercial Aspergillus PCR assays for consideration for routine service in an NHS clinical diagnostic microbiology laboratory.
Our most recent newsletters (since August 2018) can be found here.
An archive of our older newsletters (dating back to 2006) can be found here.